Keywords

Pneumonia. Mechanical ventilation. Tracheal aspirate.

Abstract

Objective: To study the validity of quantitative cultures of tracheal aspirate (TA) in comparison with the plugged telescoping catheter (PTC) for the diagnosis of mechanical ventilator-associated pneumonia. Method: Prospective multicenter study enrolling patients undergoing mechanical ventilation for longer than 72 hours. TA samples were collected from patients with suspected ventilator-associated pneumonia, followed by PTC sampling. Quantitative cultures were performed on all samples. Patients were classified according to the presence or not of pneumonia, based on clinical and radiologic criteria, clinical course and autopsy findings. The cutoff points were ≥103 colony-forming units (cfu) per mL for PTC cultures; the TA cutoffs analyzed were ≥105 and ≥106 cfu/mL. Results: Of the 120 patients studied, 84 had diagnoses of pneumonia and 36 did not (controls). The sensitivity values for TA≥106, TA≥105, and PTC, respectively, were 54% (95% confidence interval [CI], 42%-64%), 71% (95% CI, 60%-81%), and 68% (95% CI, 57%-78%). The specificity values were 75% (95% CI, 58%-88%), 58% (95% CI, 41%-74%), and 75% (95% CI, 58%-88%), respectively. Staphylococcus aureus was the microorganism most frequently isolated in both TA and PTC samples, followed in frequency by Pseudomomonas aeruginosa in TA samples and Haemophilus influenzae in PTC samples. No significant differences were found between the sensitivity of TA≥105 and that of PTC, nor between the specificities of TA≥106 and PTC. Conclusions: No differences in the specificities of PTC and TA were found when a TA cutoff of ≥106 cfu/ml was used. Moreover, at a cutoff of ≥105 the sensitivity of TA was not statistically different from that of PTC. Quantitative cultures of TA can be considered acceptable for the diagnosis of ventilator-associated pneumonia.

Article

Introduction

Ventilator-associated pneumonia (VAP) is a common complication in
patients requiring mechanical ventilation. Its incidence varies
widely (9%-70%) depending on the type of population
studied1,2 and the diagnostic methods used. In recent
years, the most appropriate method for diagnosing VAP has been the
subject of growing controversy. For some time now, invasive
techniques, such as the plugged telescoping catheter (PTC) and
bronchoalveolar lavage via fiberoptic bronchoscopy have been
mentioned in the literature as the most valid techniques for the
diagnosis of this condition.3 These methods are,
however, invasive, expensive, and not exempt from complications.
Moreover, they require the use of an invasive procedure not always
available in intensive care units. Some authors have indicated that
quantitative cultures of tracheal aspirate (TA) are of equal
diagnostic value to the invasive techniques.4,5 The
current situation is, therefore, that the scientific community has
not yet agreed upon the best routine technique for the diagnosis of
VAP.6­-11 The objective of this study, proposed by
the Infectious Diseases Work Group of the Spanish Society of
Intensive and Critical Care Medicine and Coronary Units
(GTEI-SEMICYUC), was to investigate the validity of quantitative
cultures of TA and compare that method with another conventional
method generally considered valid, such as PTC, in patients with
suspected VAP.

Materials and Methods

Patient Selection

This
prospective, multicenter study enrolled patients undergoing
mechanical ventilation for more than 72 hours who were admitted
over the period of one year to the intensive care units of the 10
participating hospitals: Hospital Clínic and Hospital del
Mar in Barcelona, Hospital de Navarra, Hospital de la Princesa in
Madrid, Hospital Río Hortega in Valladolid, Hospital Valme
and Hospital Duque del Infantado in Seville, Hospital Son Dureta in
Palma de Mallorca, Hospital Parc Taulí in Sabadell, and
Hospital Conxo in Santiago de Compostela. The following variables
were recorded for each patient: number of days on mechanical
ventilation at the time of the study, underlying disease, white
blood cell count, positive end-expiratory pressure, simplified
acute physiology score (SAPS),12 clinical pulmonary
infection score (CPIS)13 for VAP, presence of acute
respiratory distress syndrome, antibiotics taken during the week
prior to the study (number and type), and total duration of
antibiotic therapy. Quantitative TA and PTC were performed in
patients suspected of having VAP on the basis of the clinical
criteria listed below. The diagnostic procedures to be carried out
(TA and PTC) were explained to all patients or to their close
family members, and informed consent was requested. The procedures
were performed in the same way in all the hospitals: TA first, and
then PTC.

Study Protocol

TA specimens
were obtained by sterile means using an aspiration catheter and
were placed in sterile containers. Then, without interrupting
mechanical ventilation, the fiberoptic bronchoscope was passed
through the endotracheal tube via a special adaptor using the
standard technique described in the literature,3,14 and
without aspirating through the internal channel prior to collection
of respiratory secretions. Local anesthetics were not used. Then
the PTC sample was collected from the area of maximum local
inflammation and purulence using the procedure described by
Wimberley et al.15 In the case of diffuse infiltrate,
the specimen was taken from the right lower lobe. Specimens were
dispatched immediately to the laboratory for bacteriological
processing. Two blood samples for culture were extracted prior to
the above procedure.

Diagnostic Criteria

Suspicion of VAP
was based on the presence of a new and persistent pulmonary
infiltrate, together with clinical signs indicative of lower
respiratory tract infection (fever ≥38°C or hypothermia of
≤35°C, increase in the volume and/or purulence of
respiratory secretions), and laboratory findings (leukocytosis
≥12 000/µL or leukopenia ≤4000/µL). The following
criteria were used to confirm the diagnosis of pneumonia: clinical
response to appropriate antibiotic treatment, absence of an
alternative diagnosis during follow up, cavitation of an
infiltrate, or autopsy data demonstrating the existence of
pneumonia. Pneumonia was considered not to be present in patients
for whom an alternative diagnosis emerged during follow up and who
did not require subsequent antibiotic treatment, and in patients
whose antibiotic treatment was withdrawn when the absence of
pneumonia was confirmed. These patients were included in the
control group. Patients not suspected of having pneumonia and in
whom no radiologic evidence of infiltrate was observed were also
included as controls.

Microbiological Processing

All the samples
obtained were cultured quantitatively using serial dilutions. The
TA samples were mechanically homogenized using glass beads and
vortexed for one minute. Serial dilutions of the TA were prepared
with normal sterile saline solution in the proportion 1:1. The
dilutions were then inoculated in various agar culture media and
were incubated at 37ºC in aerobic conditions. The personnel in
the microbiology laboratory were not aware of the diagnosis of the
patients who produced the specimens, but they were aware of the
type of sample delivered in each case.

All
microorganisms isolated were identified by standard laboratory
methods. Bacterial pathogens in concentrations of
≥103 cfu per mL in cultures of PTC specimens, and
≥106 cfu/mL in cultures of TA were considered to be
causative agents of VAP. The growth of microorganisms that are
ordinarily not pathogenic, such as Streptococcus viridans
and Staphylococcus epidermidis, were in general considered
to be colonization.

The
identification of Legionella spp, mycobacteria or
Aspergillus spp was considered diagnostic irrespective of the
colony count obtained. Isolations of Candida spp were only
considered significant when associated with positive blood
cultures, or when histological evidence of pneumonia caused by
Candida was present.

Statistical Analysis

The data were
analyzed with the statistical software package SPSS for Microsoft
Windows, version 6 (Chicago, Illinois, USA).

Quantitative
variables are expressed as means (SD), and qualitative variables as
the frequency of distribution of each one of the categories. Means
were compared the Student t test or Mann-Whitney U-test
depending on whether or not the distribution of the variable was
normal.

Causative
variables were compared using the χ² test. When any of the
expected values in a 2x2 table were below 5, the Fisher exact test
was used.

Sensitivity and
specificity were calculated for a specified 95% CI.

The sensitivity
and specificity of the different methods were compared (separately
for patients with and without pneumonia) using the McNemar
test.

The level of
statistical significance accepted was 95%
(P<.05).

Results

A total of 120
cases were studied, of which 84 were diagnosed with pneumonia and
36 were not (control group). Of the 36 cases without pneumonia, 25
had some kind of radiologic infiltrate. No significant differences
were found between the baseline variables of the patients with and
without pneumonia except for CPIS and SAPS II scores and the
duration of antibiotic treatment, which were all significantly
higher in the group with pneumonia (Table 1).

The organism
most often isolated both by TA and PTC was Staphylococcus
aureus, followed by Pseudomonas aeruginosa and
Acinetobacter baumannii by TA, and Haemophilus
influenzae and P aeruginosa by PTC. Table 2 shows the
different organisms isolated using the 2 diagnostic methods in
patients with pneumonia, and their frequency.

On 3 occasions
primary pathogens were detected in the TA specimens that were not
isolated in those obtained by PTC: Aspergillus spp,
Mycobacterium tuberculosis and Legionella
pneumophila.

Table 3 shows
the number of positive cultures and the total number of
microorganisms isolated with each technique in the 2 groups
studied. In most cultures, growth of microorganisms was obtained
independently of the quantification value and technique
used.

Table 4 shows
the diagnostic yield for TA at 2 different cutoff points for
bacterial counts (≥105 and ≥106), and
for PTC. The sensitivity of TA with a cutoff of ≥105
and that of PTC were significantly higher than that of TA with a
cutoff of ≥106 (P=.0001 and .01,
respectively), and the difference between TA≥105 and
PTC≥103 was not significant (P=.40). The kappa
(κ
)
statistic for the agreement between PTC≥103 and
TA≥106 was 0.41, and the same statistic between
PTC≥103 and TA≥105 was 0.63.

Comparison of
the specificities of the tests gave the following results: the
specificity of TA≥106 and PTC≥103 was
significantly higher than that of TA≥105
(P=.014), but no significant difference was found between
TA≥106 and PTC≥103 (P=1.00).
The κ
statistic for the agreement
between TA≥106 and PTC≥103 was 0.70,
and κ
between TA≥105 and PTC≥103 was
0.63.

Of a total of 84
cases of pneumonia, concordant isolations (both tests positive for
the same microorganism or both tests negative) were produced with
both TA and PTC in 71 cases (83.8%), and the results did not agree
in 13 cases (16.2%) (Table 5). The analysis of the association
between the quantitative cultures of microorganisms that were
detected with both TA≥105 and PTC produced a
correlation coefficient (r) of 0.139 (P=.329).

Discussion

The most
noteworthy finding in this study was that, when a cutoff point of
106 cfu/mL or higher was used for TA in the diagnosis of
VAP, reasonable specificity was obtained (>70%), similar to that
obtained with PTC, although the sensitivity of TA at
≥106 was lower than that of both PTC and TA with a
cutoff of ≥105 cfu/mL. When the cutoff of TA was
changed to ≥105, the sensitivity did not differ
significantly from that of PTC. These results show an acceptable
yield for the clinical use of quantitative cultures of TA in the
diagnosis of VAP, making it an alternative to bronchoscopic
methods. Our findings are consistent with those of previous
studies, which mention a cutoff point of ≥106 cfu/mL
used to differentiate between colonization and infection with
TA.5 In 2 earlier studies, sensitivity and specificity
values of ≥70% (70% and 72%, respectively) were obtained using a
cutoff of 105 cfu/mL, and the results of the present
study confirm those findings.4,16

It is known that
both PTC and bronchoalveolar lavage are valid methods for the
diagnosis of VAP.1,17 On the other hand, the currently
most widely held opinion is that the use of qualitative cultures of
TA is not a very specific diagnostic method owing to the airway
colonization found in this type of patient. Although some groups
still continue to use qualitative cultures of TA in the diagnosis
of patients with suspected VAP, few doubts remain about the low
specificity of the technique. Two earlier studies, carried out by
Torres et al3 and Chastre et al,14 which
evaluated the methods used to diagnose lower respiratory tract
infection in patients on mechanical ventilation, found
specificities as low as 14% and 29%, respectively.

In recent years,
various investigators have tried to improve bronchoscopic
procedures for the diagnosis of VAP. In a relatively surprising
manner some of them have achieved acceptable results using methods
that do not involve obtaining samples from the area close to the
inflamed site of the infection. Blind sampling using
bronchoalveolar lavage (protected and unprotected) or using PTC has
been shown to produce a high diagnostic yield given the multifocal
and dynamic nature of VAP.18-22

On the other
hand, quantitative analysis of TA has been recommended as a simple
and useful procedure in the diagnosis of VAP,4,7,23 and
has the added advantages of being a noninvasive and more
inexpensive technique. This study showed low sensitivity and
acceptable specificity and a positive predictive value for TA with
a cutoff of ≥106 in the diagnosis of VAP. Sensitivity
improved with a cutoff of ≥105. PTC, on the other
hand, showed better results in sensitivity and in positive
predictive value. The specificity of PTC was the same as that of TA
at a cutoff of ≥106.

Reasonable
agreement was found in this study between the results from TA and
PTC samples. The same microorganism was obtained in 51 positive
cultures from 84 patients with pneumonia (60%), and 20 negative
concordant results were obtained (23.8%). Overall agreement was
83%. In only 2 cases (2.3%) were both tests positive but with
different microorganisms (Table 5).

Papazian et
al24 compared PTC and TA in 35 mechanically-ventilated
patients with bronchopneumonia, and reported a 93% agreement
between the microorganisms cultured with the two techniques.
However, the quantitative correlation between cultures obtained
using the different techniques was not high.

One of the
aspects of VAP diagnosis that has given rise to most debate is the
role played by invasive and noninvasive methods in the prognosis of
these patients and the antibiotic therapy administered. The debate
still continues, but some aspects have become clearer. When only
randomized clinical trials are analyzed, we find that
Sánchez-Nieto et al25 in a pilot study of 51
patients observed that bronchoscopic methods gave rise to more
changes in initial antibiotic treatment (42% as opposed to 16%;
P<.05), although no significant differences in mortality,
either global or attributable, or in morbidity were observed. A
limitation of that study was its small sample size and the absence
of a standard treatment protocol in the invasive group. A study
carried out by Solé Violan et al26 demonstrated
that while antibiotic therapy was modified more often based on
information from invasive techniques, the changes had no clear
effect on mortality, length of stay in the intensive care unit, or
duration of mechanical ventilation. Ruiz et al27
compared 76 patients with suspected VAP (39 noninvasive and 37
invasive) and concluded that the diagnostic yield for VAP was
similar with both techniques, as was mortality at 30 days, number
of days on mechanical ventilation, and length of stay in the
intensive care unit. The cost of the invasive investigation was
obviously higher.

A study
published by Fagon et al28 has reported positive results
with respect to the reduction of mortality on day 14, lower
sequential organ failure assessment scores on days 3 and 7, a
reduction in the use of antibiotics, and more days free of
antibiotics with the invasive technique. The limitation of this
study was that qualitative cultures of TA were used, thereby
limiting comparison with the other studies.

The results
obtained using quantitative cultures of TA and a threshold of
≥106 cfu/mL as the differentiator between
colonization and infection can be considered relatively acceptable
as part of the clinical approach to patients with suspected VAP
when the possibility of using bronchoscopic techniques is not
available, even though overall they are not as satisfactory as the
results obtained with PTC.